Events

Title	Abstract
"A Proteomics Approach to Understanding Protein Ubiquitination" by Junmin Peng, Harvard Medical School Junmin_peng@hms.harvard.edu	Ubiquitin (Ub) plays an essential role as a signal molecule for protein degradation in eukaryotes and as a regulator of protein activity and localization. Here we describe a proteomics approach for the systematic identification and characterization of Ub-conjugates from S. cerevisiae Ub-conjugates from a strain with 6xHis-tagged ubiquitin were isolated, proteolyzed, and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry for amino acid sequence determination. A total of 1,051 proteins were unambiguously identified. In addition, for 70 proteins we detected the precise site of ubiquitination (101 sites) with 18 proteins exhibiting more than one affected lysine residue. Moreover, ubiquitin itself was found to be modified at 6 sites (polyubiquitin chain) with an order of abundance: K48 > K63 > K11 >> K6, K27 and K33. Quantification experiment revealed the K48-linked chain was about as 4 times abundant as the K63-linked. Finally, 19 proteins (including ubiquitin itself) were identified as being modified by both ubiquitination and phosphorylation. The method provides a useful tool for large-scale analysis and characterization of protein ubiquitination.
"ProteoSep: A Viable All-Liquid Phase Alternative To 2D Gels" by Timothy Barder, Eprogen tbarder@eprogen.com	Eprogen, Inc. introduces ProteoSep (a novel protein mapping technology and a new and powerful way to analyze complex protein mixtures. ProteoSep is an all-liquid phase alternative to 2D PAGE that uses standard HPLC instrument technology to produce high resolution 2D maps of complex protein systems such as whole cell lysates, plasma and sera. Eprogen has developed a unique high performance chromatofocusing (CF) column to provide the pI information biologists need in the first dimension analysis of complex protein systems. The use of HPLC provides these pI fractions in the liquid phase containing the intact proteins. Subsequent analysis of these pI fractions with Eprogens proprietary reverse phase NPS(r) columns provides for the second dimension separation information based on hydrophobicity or MW, depending on whether you use UV or MS detection or both. A 2D protein map is produced using Eprogens new ProteoSep Software Suite, which provides either UV/pI or Mass/pI maps or both, displaying the proteins present in bands like that presented in a 2D PAGE gel. The all liquid-phase format provides for easy collection of sample fractions either at the pI stage or after the NPS analysis using conventional automation and multi-well plate fraction collectors and autosamplers without the need for complicated gel extractions.
"Proteome Scale Purification of Human Proteins from Bacteria" by Pascal Braun, Harvard Medical School pascal_braun@hms.harvard.edu	The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. To develop methods for the high-throughput purification of human proteins for use in biochemical assays, a test set of 32 sequence-verified human genes of varying sizes and activities was employed. Using recombinational cloning, these 32 proteins were attached to four different affinity-purification tags: hexa-histidine (His6-), calmodulin-binding peptide (CBP-), glutathione-S-transferase (GST-), and maltose-binding protein (MBP). By means of an automatable 2hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity and losses. Under denaturing conditions using the His6-tag, 84% of samples could be purified successfully as judged by a band of the correct size on an SDS-PAGE. Under non-denaturing conditions, both the GST- or MBP- tags were successful in 81% of samples, two of which were confirmed to be biochemically active. The developed methods were applied to a larger set of several hundred different and randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under non-denaturing conditions. A relational database, FLEXProt, was built to compare properties of successfully purified and failed proteins. We found that ras- and actin-like proteins, kinases, SH2- and SH3-proteins were highly successful in our conditions.